Hepatocyte organoids (HOs) hold significant potential for constructing bioartificial liver construction, toxicology research, and liver failure therapies. However, challenges such as difficulties in induced pluripotent stem cells (iPSCs) harvest and differentiation, safety concerns of tumor-derived matrices, and limited primary cell regulation hinder clinical applications. In this study, we developed a non-tumor-derived decellularized extracellular matrix (dECM) system with tunable mechanical properties and viscoelasticity to enhance stem cell proliferation and organoid functionality using thrombospondin-1 (THBS1). Nanoindentation and transcriptomic analysis revealed that THBS1 mediates adaptation and remodeling between organoids and ECM proteins, exhibiting native tissue-like viscoelasticity and up-regulated reprogramming transcriptional factors KLF4 and SOX2 via the YAP/TAZ pathway. Transplanting HOs presenting reprogramming effects into a 70 % hepatectomy model demonstrated improved liver regeneration, underscoring the potential of the THBS1-based dynamic ECM system in organoids manipulation and liver regeneration.

The biliary system is crucial for liver function, regulating bile production, secretion, and transport. Dysfunctions within this system can lead to various diseases, such as cholangiopathies and biliary fibrosis, which may progress from benign to malignant states like cholangiocarcinoma. While liver organoid research is well-established and technologically advanced, bile duct organoids (BDOs) offer significant potential. BDOs can accurately simulate the physiological structure and function of bile ducts, making them valuable tools for in-vitro biliary disease research. Here, we review the development of BDO models, focusing on stem cell-derived organoids and tissue-derived organoids. We also illustrate the role of cultivation strategies and extracellular scaffolds in supporting organoid growth and stability, including the influence of cellular components of the microenvironment and physicochemical factors. Furthermore, we discuss the applications of BDOs in biliary development, disease modeling, regenerative medicine, and drug screening. Additionally, we emphasize the transformative potential in BDO biobanks and personalized medicine, which helps to pave the way for innovative therapeutic strategies and personalized medicine. Finally, we summarize the current and prospective advancements in BDO technologies, highlighting the integration of emerging technologies such as artificial intelligence, 3D bioprinting, and organoid-on-chip systems. These technologies hold great promise for significantly enhancing both clinical and research applications in the field of biliary diseases.

Machine perfusion preservation of grafts has become the gold standard organ preservation method. It has been developed to improve the quality of grafts due to the increasing gap between demand and supply of organs for transplantation. Following successful long-term machine perfusion preservation with automated commercial devices developed for preservation of organs for transplantation, there is increasing interest in utilizing perfused discarded human organs and xenografts for a variety of purposes beyond transplantation including gene therapy and modulation, drug testing, chemotherapy, ex vivo surgery, organ supporting systems, bioengineering, and surgical training and education. Here, we review all current and potential applications of machine perfusion preservation.

Organoids and organs-on-chips (OoCs) are rapidly evolving technologies for creating miniature human tissue models. They can mimic complex physiological functions and pathological conditions, offering more realistic platforms for disease modeling, drug screening, precision medicine, and regenerative therapies. The passing of the FDA Modernization Act 2.0 has reduced animal testing requirements for drug trials, marking a significant milestone in using advanced in vitro models such as organoids and OoCs for therapeutic discovery. Apart from technical and ethical challenges, regulatory issues persist in ensuring the reliability, scientificity, and applicability of these models in drug development. This perspective explores the concept, advancements, pros and cons, and applications of organoids and OoCs, particularly in drug research and development. It also examines global regulatory agencies' policies and actions on using these models in drug evaluation, aiming to guide industry standard setting and advance regulatory science.

Injured or reactive biliary epithelial cells participate in most chronic liver injuries in a process referred to as ductular reaction, which involves multicellular interactions with marked local infiltration of macrophages and fibrogenic cell activation. The direct roles of biliary epithelial cells in shaping their cellular niche remain unknown.

We aimed at investigating the effects of biliary epithelial cell-derived acute phase response protein orosomucoid 2 (ORM2) in shaping monocyte/macrophage response to liver injury.

Transcriptome data sets from human and mouse livers were used, results were confirmed with multiplex immunofluorescence. A multicellular biliary-niche-on-a-chip derived from primary liver and blood cells (wild-type, Mdr2 -/- mice) was established to model ductular reaction. Human blood cells collected from healthy donors and intrahepatic cholangiocyte organoids derived from normal and cirrhotic liver patients were used.

Our transcriptome data set and multiplex immunofluorescence analyses indicated a previously unrecognised involvement of the acute phase response protein ORM2 in ductular reactions in both human and mouse livers. ORM2 gene expression was increased in biliatresone-challenged, bile acid-challenged and acetaminophen-challenged cholangiocytes. Cholangiocyte-derived ORM2 induced unique transcriptome changes and functional adaptation of liver macrophages. ORM2-activated macrophages exacerbated cholangiocyte cell stress and Orm2 expression, but also tended to promote fibrogenic activation of hepatic stellate cells. Mechanistically, ORM2 effects were mediated by an inositol 1,4,5-trisphosphate receptor type 2-dependent calcium pathway.

This study reveals a paracrine communication circuit during ductular reaction, in which reactive cholangiocyte-derived ORM2 reprogrammes liver macrophages, participating in a pathogenic remodelling of the immune biliary niche.

The TMPRSS2 cell surface protease is used by a broad range of respiratory viruses to facilitate entry into target cells. Together with ACE2, TMPRSS2 represents a key factor for SARS-CoV-2 infection, as TMPRSS2 mediates cleavage of viral spike protein, enabling direct fusion of the viral envelope with the host cell membrane. Since the start of the COVID-19 pandemic, TMPRSS2 has gained attention as a therapeutic target for protease inhibitors which would inhibit SARS-CoV-2 infection, but little is known about TMPRSS2 regulation, particularly in cell types physiologically relevant for SARS-CoV-2 infection. Here, we performed an unbiased genome-wide CRISPR-Cas9 library screen, together with a library targeted at epigenetic modifiers and transcriptional regulators, to identify cellular factors that modulate cell surface expression of TMPRSS2 in human colon epithelial cells. We find that endogenous TMPRSS2 is regulated by the Elongin BC-VHL complex and HIF transcription factors. Depletion of Elongin B or treatment of cells with PHD inhibitors resulted in downregulation of TMPRSS2 and inhibition of SARS-CoV-2 infection. We show that TMPRSS2 is still utilised by SARS-CoV-2 Omicron variants for entry into colonic epithelial cells. Our study enhances our understanding of the regulation of endogenous surface TMPRSS2 in cells physiologically relevant to SARS-CoV-2 infection.

The high failure rates in clinical drug development based on animal models highlight the urgent need for more representative human models in biomedical research. In response to this demand, organoids and organ chips were integrated for greater physiological relevance and dynamic, controlled experimental conditions. This innovative platform-the organoids-on-a-chip technology-shows great promise in disease modeling, drug discovery, and personalized medicine, attracting interest from researchers, clinicians, regulatory authorities, and industry stakeholders. This review traces the evolution from organoids to organoids-on-a-chip, driven by the necessity for advanced biological models. We summarize the applications of organoids-on-a-chip in simulating physiological and pathological phenotypes and therapeutic evaluation of this technology. This section highlights how integrating technologies from organ chips, such as microfluidic systems, mechanical stimulation, and sensor integration, optimizes organoid cell types, spatial structure, and physiological functions, thereby expanding their biomedical applications. We conclude by addressing the current challenges in the development of organoids-on-a-chip and offering insights into the prospects. The advancement of organoids-on-a-chip is poised to enhance fidelity, standardization, and scalability. Furthermore, the integration of cutting-edge technologies and interdisciplinary collaborations will be crucial for the progression of organoids-on-a-chip technology.

The ductular reaction (DR) is a dynamic adaptive cellular response within the liver, triggered by various hepatic insults and characterized by an expansion of dysmorphic biliary epithelial cells and liver progenitors. This complex response presents a dual role, playing a pivotal function in liver regeneration but, paradoxically, contributing to the progression of liver diseases, depending upon specific contextual factors and signaling pathways involved. This comprehensive review aims to offer a holistic perspective on the DR, focusing into its intricate cellular and molecular mechanisms, highlighting its pathological significance, and exploring its potential therapeutic implications. An up-to-date understanding of the DR in the context of different liver injuries is provided, analyzing its contributions to liver regeneration, inflammation, fibrosis, and ultimately carcinogenesis. Moreover, the review highlights the role of multiple microenvironmental factors, including the influence of extracellular matrix, tissue mechanics and the interplay with the intricate hepatic cell ecosystem in shaping the DR's regulation. Finally, in vitro and in vivo experimental models of the DR will be discussed, providing insights into how researchers can study and manipulate this critical cellular response. By comprehensively addressing the multifaceted nature of the DR, this review contributes to a more profound understanding of its pathophysiological role in liver diseases, thus offering potential therapeutic avenues for hepatic disorders and improving patient outcomes.

Liver transplantation is associated with a high risk of postoperative complications due to the complexity of the surgical procedure, recipient disease severity and the wide range of graft quality, which remains somewhat unpredictable. However, survival rates after transplantation continue to improve and the focus has thus turned to other clinically relevant endpoints including post-transplant complications, patient quality of life and costs. Procedures like liver transplantation offer the entire spectrum of post-surgical events, even in donor-recipient constellations deemed of low risk within recently defined benchmark criteria. The Clavien-Dindo classification and the CCI (comprehensive complication index) were established to assess postoperative morbidity and are widely utilised across surgical specialties. These scores depend on the number and grade of complications, which reflect the interventions required, and are frequently used to assess specific donor-recipient risk profiles and new approaches, such as machine perfusion. However, these scores are associated with inter-observer variability when used in practice, mainly due to the lack of uniform definitions. The concept of benchmarking was recently introduced in surgery and transplantation as a mechanism of standardising expected donor/recipient risk with outcomes within the first year after surgery. However, the management of complications differs significantly worldwide, as does the rating scale assigned to various complications. This may lead to inhomogeneous interpretation of study results, leading to difficulty in assessing the clinical effects of novel preservation technologies and other therapeutics in liver transplantation. This article critically discusses frequent challenges associated with risk and outcome assessment following liver transplantation.

Liver regeneration is supported by hepatocytes and, in certain conditions, biliary epithelial cells (BECs). BECs are facultative liver stem cells that form organoids in culture and engraft in damaged livers. However, BEC heterogeneity in the homeostatic liver remains to be fully elucidated. Here, we exploit systemic lentiviral vector (LV) administration to achieve efficient and lifelong gene transfer to BECs in mice. We find that LV-marked BECs retain organoid formation potential and predominantly respond to liver damage; however, they are less clonogenic and display a hepatocyte-primed transcriptome compared to untransduced BECs. We thus identify a BEC subset committed to hepatocyte lineage in the absence of liver damage, characterized by a transcriptional network orchestrated by hepatocyte nuclear factor 4α. We also report in vivo targeting of such BECs in non-human primates. This work highlights intrinsic BEC heterogeneity and that in vivo LV gene transfer to the liver may persist following BEC-mediated repair of hepatic damage.

The liver fulfils a plethora of vital functions and, due to their importance, liver dysfunction has life-threatening consequences. Liver disorders currently account for more than two million deaths annually worldwide and can be classified broadly into three groups, considering their onset and aetiology, as acute liver diseases, inherited metabolic disorders and chronic liver diseases. In the most advanced and severe forms leading to liver failure, liver transplantation is the only treatment available, which has many associated drawbacks, including a shortage of organ donors. Cell therapy via fully mature cell transplantation is an advantageous alternative that may be able to restore a damaged organ's functionality or serve as a bridge until regeneration can occur. Pioneering work has shown that transplanting adult hepatocytes can support liver recovery. However, primary hepatocytes cannot be grown extensively in vitro as they rapidly lose their metabolic activity. Therefore, different cell sources are currently being tested as alternatives to primary cells. Human pluripotent stem cell-derived cells, chemically induced liver progenitors, or 'liver' organoids, hold great promise for developing new cell therapies for acute and chronic liver diseases. This Review focuses on the advantages and drawbacks of distinct cell sources and the relative strategies to address different therapeutic needs in distinct liver diseases.

Organoids are stem-cell derived tissue structures mimicking specific structural and functional characteristics of human organs. Despite significant advancements in the field over the last decade, challenges like limited long-term functional culture and lack of maturation are hampering the implementation of organoids in biomedical research. Culture of organoids in microfluidic chips is being used to tackle these challenges through dynamic and precise control over the organoid microenvironment. This review highlights the significant breakthroughs that have been made in the innovative field of "organoids-on-chip," demonstrating how these have contributed to advancing organoid models. We focus on the incorporation of organoids representative for various tissues into chips and discuss the latest findings in multi-organoids-on-chip approaches. Additionally, we examine current limitations and challenges of the field towards the development of reproducible organoids-on-chip systems. Finally, we discuss the potential of organoids-on-chip technology for both in vitro and in vivo applications.

The dominant paradigm in biomedicine focuses on genetically-specified components of cells and their biochemical dynamics, emphasizing bottom-up emergence of complexity. Here, I explore the biomedical implications of a complementary emerging field: diverse intelligence. Using tools from behavioral science and multiscale neuroscience, we can study development, regenerative repair, and cancer suppression as behaviors of a collective intelligence of cells navigating the spaces of possible morphologies and transcriptional and physiological states. A focus on the competencies of living material-from molecular to organismal scales-reveals a new landscape for interventions. Such top-down approaches take advantage of the memories and homeodynamic goal-seeking behavior of cells and tissues, offering the same massive advantages in biomedicine and bioengineering that reprogrammable hardware has provided information technologies. The bioelectric networks that bind individual cells toward large-scale anatomical goals are an especially tractable interface to organ-level plasticity, and tools to modulate them already exist. This suggests a research program to understand and tame the software of life for therapeutic gain by understanding the many examples of basal cognition that operate throughout living bodies.

The global shortage of suitable donor livers for transplantation has prompted efforts to expand the donor pool by using extended criteria donors. Machine preservation technology has shown promise in optimizing graft preservation and improving logistics. Additionally, it holds potential for organ repair, regeneration, therapeutic applications during extended preservation periods, and enhancing organ allocation.

We conducted a comprehensive literature review using PubMed, Embase, and Web of Science databases. All studies published between January 1, 2022, and February 7, 2024, that described machine perfusion preservation of livers for more than 24 h were eligible for inclusion. The findings were synthesized in a narrative review format to highlight key benefits and advancements.

We identified eleven studies from multiple research groups, employing various techniques, devices, and preservation durations. Perfusion durations ranged from 1 to 13 days, with notable variations in protocols for long-term preservation beyond 24 h. Viability was assessed during perfusion only. No livers were transplanted. Among the reviewed studies, the introduction of a dialysis system emerged as the most effective strategy for managing waste accumulation during long-term liver perfusion. Differences were also observed in hemodynamics, oxygenation, organ chambers, supplemental regimens, and glycemic control.

Over the past two years, substantial progress has been made in refining protocols for long-term liver machine perfusion, with significant advancements in waste management, enabling successful multi-day perfusions. While these developments are promising, further research is necessary to standardize and optimize long-term perfusion protocols, establishing a reliable platform for both organ preservation and therapeutic applications.

The biliary tract is a ductal network comprising the intrahepatic (IHBDs) and extrahepatic bile duct (EHBDs). Biliary duct disorders include cholangitis, neoplasms, and injury. However, the underlying mechanisms are not fully understood. With advancements in 3D culture technology, cholangiocyte organoids (COs) derived from primary tissues or induced pluripotent stem cells (iPSCs) can accurately replicate the structural and functional properties of biliary tissues. These organoids have become powerful tools for studying the pathogenesis of biliary diseases, such as cystic fibrosis and primary sclerosing cholangitis, and for developing new therapeutic strategies for cholangiocarcinoma. Additionally, COs have the potential to repair bile duct injuries and facilitate transplantation therapies. This review also discusses the use of organoids in genetically engineered mouse models to provide mechanistic insights into tumorigenesis and cancer progression. Continued innovation and standardization of organoid technology are crucial for advancing precision medicine for biliary diseases and cancer.

Ischemia-reperfusion injury (IRI) and bile salt toxicity are significant contributors to post-transplant cholangiopathy. Ferroptosis appears to play a critical role in intrahepatic bile duct injury induced by ischemia-reperfusion (I/R) and bile salt toxicity. Our study aimed to elucidate the role of ferroptosis in bile duct injuries and its potential as a therapeutic target for liver diseases. Mouse models of liver ischemia-reperfusion (I/R) and α-naphthyl isocyanate (ANIT)-induced liver cholestasis were employed to investigate the role of ferroptosis in intrahepatic bile duct injury in vivo. Hypoxia-reoxygenation (H/R) and bile salt treatment models were utilized to simulate the post-transplant bile duct injury process in vitro. In mouse models of liver I/R and cholestasis, we observed a downregulation of glutathione peroxidase 4 (GPX4) and an upregulation of lipid peroxidation levels in bile duct cells. Furthermore, the ferroptosis inhibitor Liproxstatin-1 (Lip-1) significantly attenuated intrahepatic bile duct injuries. Ferroptosis inhibitors alleviated cell death and lipid peroxide accumulation in human intrahepatic biliary epithelial cells (HiBECs) subjected to H/R or glycochenodeoxycholate (GCDCA) treatment. GCDCA treatment led to ferroptosis in HiBECs along with ferritin degradation. Inhibition of autophagy alleviated GCDCA-induced bile duct cell death. Our study suggested that ferroptosis played an important role of in the intrahepatic bile duct injury during I/R or cholestasis.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer-related mortality worldwide1,2. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulate progressive driver mutations3, with hepatocytes the most likely cell of origin2. However, the landscape of driver mutations in HCC is broadly independent of the underlying aetiologies4. Despite an increasing range of systemic treatment options for advanced HCC, outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations5,6. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC7. Here we generated a suite of genetically driven immunocompetent in vivo and matched in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance and metastasis. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with the human histopathology. In a proof-of-principle analysis, we verified response to standard-of-care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not previously been linked to HCC treatment. Cladribine acts in a highly effective subtype-specific manner in combination with standard-of-care therapy.

Liver diseases represent a growing global health challenge, and the increasing prevalence of obesity and metabolic disorders is set to exacerbate this crisis. To meet evolving regulatory demands, patient-specific in vitro liver models are essential for understanding disease mechanisms and developing new therapeutic approaches. Organoid models, which faithfully recapitulate liver biology, can be established from both non-malignant and malignant liver tissues, offering insight into various liver conditions, from acute injuries to chronic diseases and cancer. Improved understanding of liver microenvironments, innovative biomaterials, and advanced imaging techniques now facilitate comprehensive and unbiased data analysis, paving the way for personalised medicine. In this review, we discuss state-of-the-art patient-derived liver organoid models, recent technological advancements, and strategies to enhance their clinical impact.

Human organoids are a widely used tool in cell biology to study homeostatic processes, disease, and development. The term organoids covers a plethora of model systems from different cellular origins that each have unique features and applications but bring their own challenges. This review discusses the basic principles underlying organoids generated from pluripotent stem cells (PSCs) as well as those derived from tissue stem cells (TSCs). We consider how well PSC- and TSC-organoids mimic the different intended organs in terms of cellular complexity, maturity, functionality, and the ongoing efforts to constitute predictive complex models of in vivo situations. We discuss the advantages and limitations associated with each system to answer different biological questions including in the field of cancer and developmental biology, and with respect to implementing emerging advanced technologies, such as (spatial) -omics analyses, CRISPR screens, and high-content imaging screens. We postulate how the two fields may move forward together, integrating advantages of one to the other.

Organoid culture has emerged as a forefront technology in the life sciences field. As "in vitro micro-organs", organoids can faithfully recapitulate the organogenesis process, and conserve the key structure, physiological function and pathological state of the original tissue or organ. Consequently, it is widely used in basic and clinical studies, becoming important preclinical models for studying diseases and developing therapies. Here, we introduced the definition and advantages of organoids and described the development and advances in hepatobiliary organoids research. We focus on applying hepatobiliary organoids in benign and malignant diseases of the liver and biliary tract, drug research, and regenerative medicine to provide valuable reference information for the application of hepatobiliary organoids. Despite advances in research and treatment, hepatobiliary diseases including carcinoma, viral hepatitis, fatty liver and bile duct defects have still been conundrums of the hepatobiliary field. It is necessary and crucial to study disease mechanisms, establish efficient and accurate research models and find effective treatment strategies. The organoid culture technology shed new light on solving these issues. However, the technology is not yet mature, and many hurdles still exist that need to be overcome. The combination with new technologies such as CRISPR-HOT, organ-on-a-chip may inject new vitality into future development.

Developing disease models and screening for effective drugs are key areas of modern medical research. Traditional methodologies frequently fall short in precisely replicating the intricate architecture of bodily tissues and organs. Nevertheless, recent advancements in biomaterial-assisted organoid technology have ushered in a paradigm shift in biomedical research. This innovative approach enables the cultivation of three-dimensional cellular structures in vitro that closely emulate the structural and functional attributes of organs, offering physiologically superior models compared to conventional techniques. The evolution of biomaterials plays a pivotal role in supporting the culture and development of organ tissues, thereby facilitating more accurate disease state modeling and the rigorous evaluation of drug efficacy and safety profiles. In this review, we will explore the roles that various biomaterials play in organoid development, examine the fundamental principles and advantages of utilizing these technologies in constructing disease models, and highlight recent advances and practical applications in drug screening using disease-specific organoid models. Additionally, the challenges and future directions of organoid technology are discussed. Through continued research and innovation, we aim to make remarkable strides in disease treatment and drug development, ultimately enhancing patient quality of life.

Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.

Organoids are innovative three-dimensional and self-organizing cell cultures of various lineages that can be used to study diverse tissues and organs. Human organoids have dramatically increased our understanding of developmental and disease biology. They provide a patient-specific model to study known diseases, with advantages over animal models, and can also provide insights into emerging and future health threats related to climate change, zoonotic infections, environmental pollutants or even microgravity during space exploration. Furthermore, organoids show potential for regenerative cell therapies and organ transplantation. Still, several challenges for broad clinical application remain, including inefficiencies in initiation and expansion, increasing model complexity and difficulties with upscaling clinical-grade cultures and developing more organ-specific human tissue microenvironments. To achieve the full potential of organoid technology, interdisciplinary efforts are needed, integrating advances from biology, bioengineering, computational science, ethics and clinical research. In this Review, we showcase pivotal achievements in epithelial organoid research and technologies and provide an outlook for the future of organoids in advancing human health and medicine.

In recent years, there has been resurgence in donation after circulatory death (DCD). Despite that, the number of organs transplanted from these donors remains low due to concerns about their function and a lack of an objective assessment at the time of donation. This overview examines the current DCD practices and the classification modifications to accommodate regional perspectives. Several risk factors underscore the reluctance to accept DCD organs, and we discuss the modern strategies to mitigate them. The advent of machine perfusion technology has revolutionized the field of DCD transplantation, leading to improved outcomes and better organ usage. With many strategies at our disposal, there is an urgent need for comparative trials to determine the optimal use of perfusion technologies for each donated organ type. Additional progress in defining therapeutic strategies to repair the damage sustained during the dying process should further improve DCD organ utilization and outcomes. However, there remains wide variability in access to DCD donation and transplantation, and organizational efforts should be doubled up with consensus on key ethical issues that still surround DCD donation in the era of machine perfusion.

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

The liver is a vital organ responsible for numerous metabolic processes in the human body, including the metabolism of drugs and nutrients. After liver damage, the organ can rapidly return to its original size if the causative factor is promptly eliminated. However, when the harmful stimulus persists, the liver's regenerative capacity becomes compromised. Substantial theoretical feasibility has been demonstrated at the levels of gene expression, molecular interactions, and intercellular dynamics, complemented by numerous successful animal studies. However, a robust model and carrier that closely resemble human physiology are still lacking for translating these theories into practice. The potential for liver regeneration has been a central focus of ongoing research. Over the past decade, the advent of organoid technology has provided improved models and materials for advancing research efforts. Liver organoid technology represents a novel in vitro culture system. After several years of refinement, human liver organoids can now accurately replicate the liver's morphological structure, nutrient and drug metabolism, gene expression, and secretory functions, providing a robust model for liver disease research. Regenerative medicine aims to replicate human organ or tissue functions to repair or replace damaged tissues, restore their structure or function, or stimulate the regeneration of tissues or organs within the body. Liver organoids possess the same structure and function as liver tissue, offering the potential to serve as a viable replacement for the liver, aligning with the goals of regenerative medicine. This review examines the role of liver organoids in regenerative medicine.

The limited replicative potential of primary hepatocytes (Hep) is a major hurdle for obtaining sufficient quantity and quality hepatocytes during cell therapy in patients with liver failure. Intrahepatic cholangiocyte organoids (ICOs) derived from intrahepatic bile ducts differentiate into both hepatocytes and cholangiocytes in vitro. Here, we studied in vivo effects of transplanting ICOs and Hep in chronic liver injury mice models. Well characterized primary mouse ICOs and Hep were mixed in decellularized liver (DCL) matrix hydrogels and injected into the subcapsular left lateral liver lobe of CCl4-induced liver injury models whereas mice given DCL alone were in the sham group. Two weeks post-transplantation, transplanted liver lobes were collected and studied by histology and RNA sequencing. Transplanted animals did not exhibit any tumors, mortality or morbidity. Mice livers transplanted with ICOs had increased cellular proliferation and vascularization as compared to Hep transplanted mice or sham. Collagen deposition in the liver was significantly reduced and serum albumin levels were significantly increased in transplanted groups compared to the sham group. Expression of genes associated with hepatocyte differentiation was highest in Hep transplanted livers among the three groups, but they were also upregulated in ICO transplanted livers compared to sham. Our study demonstrates that ICOs encapsulated in DCL hydrogels when transplanted in chronically injured mice livers engraft well and show hepatocyte differentiation and reduction of fibrosis, indicating that hydrogel transplanted cholangiocyte organoids may serve as an efficient cell source and therapy for renewal of hepatocytes, restoration of hepatocyte functions and resolution of liver injury.

Chronic hepatobiliary damage progressively leads to fibrosis, which may evolve into cirrhosis and/or hepatocellular carcinoma. The fight against the increasing incidence of liver-related morbidity and mortality is challenged by a lack of clinically validated early-stage biomarkers and the limited availability of effective anti-fibrotic therapies. Current research is focused on uncovering the pathogenetic mechanisms that drive liver fibrosis. Drugs targeting molecular pathways involved in chronic hepatobiliary diseases, such as inflammation, hepatic stellate cell activation and proliferation, and extracellular matrix production, are being developed. Etiology-specific treatments, such as those for hepatitis B and C viruses, are already in clinical use, and efforts to develop new, targeted therapies for other chronic hepatobiliary diseases are ongoing. In this review, we highlight the major molecular changes occurring in patients affected by metabolic dysfunction-associated steatotic liver disease, viral hepatitis (Delta virus), and autoimmune chronic liver diseases (autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis). Further, we describe how this knowledge is linked to current molecular therapies as well as ongoing preclinical and clinical research on novel targeting strategies, including nucleic acid-, mesenchymal stromal/stem cell-, and extracellular vesicle-based options. Much clinical development is obviously still missing, but the plethora of promising potential treatment strategies in chronic hepatobiliary diseases holds promise for a future reversal of the current increase in morbidity and mortality in this group of patients.

Biliary duct injury, biliary atresia (BA), biliary tract tumors, primary sclerosing cholangitis (PSC), and other diseases are commonly encountered in clinical practice within the digestive system. To gain a better understanding of the pathogenesis and development of these diseases and explore more effective treatment methods, organoid technology has recently garnered significant attention. Organoids are three-dimensional structures derived from stem/progenitor cells that can faithfully mimic the intricate structure and physiological function of tissues or organs in vitro. They provide a valuable platform for studying the pathogenesis of biliary tract diseases and offer novel possibilities for repairing and regenerating biliary tract injuries. The main seed cells used to construct biliary tract organoids include primary human biliary tract epithelial cells as well as pluripotent stem cells. The construction of these organoids involves various techniques such as traditional embedding technology, rotary culture technology, hanging drop culture technology, along with emerging approaches like organ chip technology, three-dimensional (3D) printing technology, and four-dimensional (4D) printing technology. This article comprehensively reviews the construction methods of biliary tract organoids while discussing their applications in disease modeling research on disease mechanisms drug screening tissue/organ repair; it also highlights current challenges and suggests future research directions regarding biliary tract organoids which will serve as references for treating common refractory digestive system diseases in clinical practice.

Stem cells (SCs) can self-replicate and differentiate into multiple lineages. Organoids, 3D cultures derived from SCs, can replicate the spatial structure and physiological characteristics of organs in vitro. Skin organoids can effectively simulate the physiological structure and function of skin tissue, reliably restoring the natural skin ecology in various in vitro environments. Skin organoids have been employed extensively in skin development and pathology research, offering valuable insights for drug screening. Moreover, they play crucial roles in skin regeneration and tissue repair. This in-depth review explores the construction and applications of skin organoids in wound healing, with a focus on their construction process, including skin appendage integration, and significant advancements in wound-healing research.

Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.

Bile duct regeneration is hypothesized to prevent biliary strictures, a leading cause of morbidity after liver transplantation. Assessing the capacity for biliary regeneration may identify grafts as suitable for transplantation that are currently declined, but this has been unfeasible until now. This study used long-term ex situ normothermic machine perfusion (LT-NMP) to assess biliary regeneration. Human livers that were declined for transplantation were perfused at 36 °C for up to 13.5 days. Bile duct biopsies, bile, and perfusate were collected throughout perfusion, which were examined for features of injury and regeneration. Biliary regeneration was defined as new Ki-67-positive biliary epithelium following severe injury. Ten livers were perfused for a median duration of 7.5 days. Severe bile duct injury occurred in all grafts, and biliary regeneration occurred in 70% of grafts. Traditional biomarkers of biliary viability such as bile glucose improved during perfusion but this was not associated with biliary regeneration (P > .05). In contrast, the maintenance of interleukin-6 and vascular endothelial growth factor-A levels in bile was associated with biliary regeneration (P = .017 for both cytokines). This is the first study to demonstrate biliary regeneration during LT-NMP and identify a cytokine signature in bile as a novel biomarker for biliary regeneration during LT-NMP.

Biohybrid regenerative bioelectronics are an emerging technology combining implantable devices with cell transplantation. Once implanted, biohybrid regenerative devices integrate with host tissue. The combination of transplant and device provides an avenue to both replace damaged or dysfunctional tissue, and monitor or control its function with high precision. While early challenges in the fusion of the biological and technological components limited development of biohybrid regenerative technologies, progress in the field has resulted in a rapidly increasing number of applications. In this perspective the great potential of this emerging technology for the delivery of therapy is discussed, including both recent research progress and potential new directions. Then the technology barriers are discussed that will need to be addressed to unlock the full potential of biohybrid regenerative devices.

Kidney disease has become a growing public health problem worldwide, and there is an urgent need to develop reliable models for investigating novel and effective treatment strategies. In recent years, kidney organoids, as novel models different from traditional two-dimensional cells and model animals, have attracted more and more attention. Current advances have allowed the generation of kidney organoids from the directed differentiation of induced pluripotent stem cells (iPSCs), which possess similar characteristics to embryonic stem cells, but bypass ethical constraints and have a wide range of sources.

Herein, the methods of generating renal organoids from iPSCs, the applications of iPSC-derived renal organoids in disease modeling, drug effectiveness detection, and regenerative medicine as well as the challenges were reviewed.

iPSC-derived renal organoids can be used to model kidney diseases and are great models for studying kidney injury and toxicity. Many efforts are needed to finally apply organoids into clinical application.

Severe damage to the intrahepatic biliary duct (IHBD) network occurs in multiple human advanced cholangiopathies, such as primary sclerosing cholangitis, biliary atresia and end-stage primary biliary cholangitis. Whether and how a severely damaged IHBD network could reconstruct has remained unclear. Here we show that, although the gallbladder is not directly connected to the IHBD, there is a common hepatic duct (CHD) in between, and severe damage to the IHBD network induces migration of gallbladder smooth muscle cells (SMCs) to coat the CHD in mouse and zebrafish models. These gallbladder-derived, CHD-coating SMCs produce retinoic acid to activate Sox9b in the CHD, which drives proliferation and ingrowth of CHD cells into the inner liver to reconstruct the IHBD network. This study reveals a hitherto unappreciated function of the gallbladder in the recovery of injured liver, and characterizes mechanisms involved in how the gallbladder and liver communicate through inter-organ cell migration to drive tissue regeneration. Carrying out cholecystectomy will thus cause previously unexpected impairments to liver health.

Fibrolamellar Hepatocellular Carcinoma (FLC) is a rare liver cancer characterized by a fusion oncokinase of the genes DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). A few FLC-like tumors have been reported showing other alterations involving PKA. To better understand FLC pathogenesis and the relationships among FLC, FLC-like, and other liver tumors, we performed a massive multi-omics analysis. RNA-seq data of 1412 liver tumors from FLC, hepatocellular carcinoma, hepatoblastoma and intrahepatic cholangiocarcinoma are analyzed, obtaining transcriptomic signatures unrestricted by experimental processing methods. These signatures reveal which dysregulations are unique to specific tumors and which are common to all liver cancers. Moreover, the transcriptomic FLC signature identifies a unifying phenotype for all FLC tumors regardless of how PKA was activated. We study this signature at multi-omics and single-cell levels in the first spatial transcriptomic characterization of FLC, identifying the contribution of tumor, normal, stromal, and infiltrating immune cells. Additionally, we study FLC metastases, finding small differences from the primary tumors.

Liver transplantation represents a pivotal intervention in the management of end-stage liver disease, offering a lifeline to countless patients. Despite significant strides in surgical techniques and organ procurement, ethical dilemmas and debates continue to underscore this life-saving procedure. Navigating the ethical terrain surrounding this complex procedure is hence paramount. Dissecting the nuances of ethical principles of justice, autonomy and beneficence that underpin transplant protocols worldwide, we explore the modern challenges that plaques the world of liver transplantation. We investigate the ethical dimensions of organ transplantation, focusing on allocation, emerging technologies, and decision-making processes. PubMed, Scopus, Web of Science, Embase and Central were searched from database inception to February 29, 2024 using the following keywords: "liver transplant", "transplantation", "liver donation", "liver recipient", "organ donation" and "ethics". Information from relevant articles surrounding ethical discussions in the realm of liver transplantation, especially with regards to organ recipients and allocation, organ donation, transplant tourism, new age technologies and developments, were extracted. From the definition of death to the long term follow up of organ recipients, liver transplantation has many ethical quandaries. With new transplant techniques, societal acceptance and perceptions also play a pivotal role. Cultural, religious and regional factors including but not limited to beliefs, wealth and accessibility are extremely influential in public attitudes towards donation, xenotransplantation, stem cell research, and adopting artificial intelligence. Understanding and addressing these perspectives whilst upholding bioethical principles is essential to ensure just distribution and fair allocation of resources. Robust regulatory oversight for ethical sourcing of organs, ensuring good patient selection and transplant techniques, and high-quality long-term surveillance to mitigate risks is essential. Efforts to promote equitable access to transplantation as well as prioritizing patients with true needs are essential to address disparities. In conclusion, liver transplantation is often the beacon of hope for individuals suffering from end-stage liver disease and improves quality of life. The ethics related to transplantation are complex and multifaceted, considering not just the donor and the recipient, but also the society as a whole.